The rapid and accurate determination of specific metabolites present in biofluids is a very demanding task which is essential in both medicine and chemistry. L-carnitine (3-hydroxy-4-N-trimethylammonium butyrate) is an important metabolite which participates in a series of biological paths and therefore its determination is of diagnostic importance. A single quantum coherence filtering (1)H NMR methodology was used for the accurate and rapid determination of L-carnitine in human serum samples. The methodology is based on spectral simplification, and specifically on the distinction of the N-methyl proton signal of L-carnitine that is greatly overlapped in the (1)H-NMR spectrum of serum. The quantitative results provided by the proposed method are in excellent agreement with those obtained by the enzymatic method, which is widely used. The proposed method is rapid (~20 min of experimental time), selective, sensitive, and has good analytical characteristics (accuracy, reproducibility). Selected protein precipitation methods were also investigated and sample pretreatment with EtOH is suggested.